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Detection of Serum Antibody by the Antimitogen Assay Against Streptococcal Erythrogenic Toxins

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Detection of Serum Antibody by the Antimitogen Assay Against Streptococcal Erythrogenic Toxins 003 1-3998/90/2701-00 1 1$02.00/0 PEDIATRIC RESEARCH Vol. 27. No. I. 1990 Copyright O 1990 International Pediatric Research Foundation, Inc Printed in U.S. A Detection of Serum Antibody by the Antimitogen Assay against Streptococcal Erythrogenic Toxins. Age Distribution in Children and the Relation to Kawasaki Disease YOSHIAKI ABE, SHOKO NAKANO, TAKAKO NAKAHARA, YUKIO KAMEZAWA, IWAO KATO, HIROHARU USHIJIMA, KAZUYA YOSHINO, SHIGERU ITO, SEIJI NOMA, SHOKO OKITSU, AND MASAKO TAJIMA Depurtlnen1.s qf Puthology [Y.A.,S.N.],Pediatrics [H. U., K. Y., S I., S.N., S.O.1, and Central Clinical Laboratory [M. T.], Teilcyo University School qf Medicme, Tokyo Department of Microbiology [T.N., Y.K.], Saitamu College (~'Heal~h,Uuawa; and Department of Micuobiology [I.K.], Facility qfMediciize, Chibu Lrniversity, Chiha, Japan ABSTRACT. We describe a new method to measure hu- been reported also in Korea, the United States, Europe, and man serum antibody against streptococcal erythrogenic Australia (3-7). Pathology of the disease is characterized by toxins that uses inhibition of lymphocyte mitogenicity of systemic vasculitis simulating or identical to infantile periarteritis the toxins as the indicator. Sera from 53% of 53 Kawasaki nodosa (8-1 3). Coronary arteries are most severely affected. Rare disease patients contained specific inhibitory activity but sudden death is caused by myocardial infarction due to against A toxin, whereas only 15% had serum inhibitory coronary thrombosis or by rupture of a coronary aneurysm. The activity against B toxin. The specific anti-A toxin serum etiology remains unknown, although the involvement of an inhibitor was found in 10% of 118 age-matched control infectious agent is suspected on clinical and epidemiologic patients suffering from various infections and allergic dis- grounds. Many attempts have been made to find the causative eases (p = 0.001, compared to Kawasaki disease patients). microorganism among viruses, bacteria, spirochetes, and fungi, Serum inhibitory activity was detected in a small number but no definite evidence has been obtained (14). Suspicion that of patients with P-hemolytic streptococcal infection (3119) KD may be a toxin-related disease comes from several reports and in none of the age-matched healthy children (0117). indicating similarities in symptoms between toxic shock syn- However, four of seven cord blood sera samples and five drome and KD (15-18). Similarities to scarlet fever have been of 13 sera samples from healthy neonates contained the noted in skin and oral eruptions of KD, but KD affects much inhibitor, a result suggesting passive transfer from moth- younger children than scarlet fever, and isolation of P-hemolytic ers. Most of the antimitoeen-~ositivesera were also ~osi- streptococci is infrequent in patients with KD (2). However, tive by ELISA of IgG anyibody against A toxin, and^^^ some symptoms of KD are similar to those known to result from fractions of the positive sera remained positive in both SET, which play a pathogenetic role in scarlet fever. These assays. Thus, it is possible that the specific serum inhibitor biologic properties include pyrogenicity, erythrogenic activity, detected by the antimitogen assay represents anti-A toxin and lymphocyte mitogenicity (I 9,20). In our report, we describe antibody. The role of toxin-producing bacteria in the path- a new method to measure the anti-SET antibody by using the ogenesis of Kawasaki disease remains to be investigated. lymphocyte mitogenicity of the toxins as the indicator. We have (Pediatv Res 27: 11-15, 1990) found a significantly higher frequency of serum inhibitory activ- ity against erythrogenic toxin A mitogenicity in KD patients Abbreviations when compared with age-matched control children. The anti- toxin inhibitory activity detected in sera of KD patients suggests KID, Kawasaki disease the possibility that toxin-producing bacteria may contribute to SET, streptococcal erythrogenic toxins pathogenesis of this disease. TdR, thymidine Con A, concanavalin A AMA, antimotogen assay MATERIALS AND METHODS OD, outer diamer acid), BSA, carbonyl iron powder, and N-2-hydroxyethyl-piper- azine-N'-2-ethane sulfonic acid were purchased from Sigma Chemical Co., St. Louis, MO.~H-T~R,-S~act 78 ci/m~,was KD, or mucocutaneous lymph node syndrome, is an acute purchased from Amersham Japan Co., Tokyo, Japan. Other febrile infantile disease that has become known as a new clinical chemicals and reagents were Dulbecco's modified Eagle medium entity since Kawasaki's first article was published in Japanese (GIBCO, Grand Island, NY), Vectastain ABC kit (Vector Lab- about 20 y ago (1). Kawasaki et al. (2) later gave an outline of oratories, through Funakoshi Pharmaceutical Co., Tokyo, Ja- clinical and epidemiologic aspects of the disease in English (2). pan), goat antihuman IgG serum (Tago Inc., Burlingame, CA), KD has not only been extensively encountered in Japan, but has Con A, Miles-Yeda Ltd., Rehovot, Israel), protein A Sepharose (Pharmacia Japan Co., Tokyo, Japan), Tween 20 (Wako Pure Received July 11, 1988: accepted August 22, 1989. Chemical Industries, Tokyo, Japan), polyvinyl alcohol (polym- Correspondence Yoshiaki Abe, M.D., Department of Pathology, Teikyo Uni- versity School of Medicine, Kaga 2-1 1-1. Itabashi, Tokyo 173, Japan. erization degree 2000, Koso Chemical Co. Tokyo, Japan), peni- Supported in part by a grant from the Japan Heart Foundation, Tokyo. cillin and streptomycin (Meiji, Tokyo, Japan). PBS, pH 7.2,O. 15 12 ABE ET AL. M, carbonate buffer, pH 9.6, 0.05 M, citrate buffer, pH 4.0, 0.1 distributed in 0.1 mL vol in each well of 96-well plastic plate, M, and glycine-HC1 buffer, pH 3.0, 0.1 M, were prepared as sealed and kept at 4°C overnight. Following washing, procedure described (2 1) was carried out three times at the end of each step by PBS SET preparations. Purified erythrogenic toxins A and B were containing 0.05% Tween 20. BSA in PBS (1 mg/mL) for recoat- obtained from culture supernatant of NY5 strain Streptococcus ing was distributed in 0.1 mL vol in each well and placed at pyogenes from the stock of the Institute of Medical Science, the room temperature for 15 min. Twenty pL of 20-fold dilutions of University of Tokyo, Tokyo, Japan. Ammonium sulfate precip- human sera, or fractions separated by protein A Sepharose itate at 314 saturation was used as crude toxin. Further purifi- column, were then added, and incubated for another 30 min. cation was carried out as described (22). Physicochemical and After washing, BSA in PBS was refilled, incubated for 15 min, biologic characteristics of the purified A toxin are briefly as and 10 pL of a goat antihuman IgG serum were added for follows. It produced a single band on SDS-PAGE, showing M, another 30 min. The antiserum was used at 1000-fold dilution 28 000, and had isoelectric points of 5.0 (major) and 5.2 (minor). in PBS. Thereafter, each one drop (approximately 50 pL) of the HPLC revealed one sharp symmetric peak, showing a high degree reagents included in Vectastain ABC kit was successively distrib- of homogeneity. The result of amino acid analysis. of the toxin uted and incubated as above. They were normal rabbit serum, was in accordance with Gerlach et al. (23) and with Weeks and biotin-labeled anti-goat 1gG rabbit IgG, avidin-biotin complex, Ferretti (24). An antiserum raised in a rabbit immunized with and biotin-conjugated peroxidase. Finally, 0.1 mL of the sub- Freund's complete adjuvant against the purified A toxin pro- strate solution containing 0.04% H202and 0.2 mM 2-2'-azino- duced a single precipitation line in gel diffusion, specifically bis(3-ethylbenzthiazoline-6-sulfonic acid) dissolved in the 10-fold abolished erythematous skin reaction, and inhibited the lympho- diluted citrate buffer was distributed and incubated at room cyte mitogenicity in a dose-dependent fashion. B toxin had a temperature for 30 min or more. The OD was measured at 405 isoelectric point of 7.9. Specific antisera to inhibit biologic ac- nm. Wells without addition of human serum and the goat tivities of the B toxin have not been obtained. The isoelectric antihuman IgG serum served as blanks. Each test was carried points of the A and B toxins are similar to those of streptococcal out in duplicate. Mean OD of 0.20 or more was taken as positive. pyrogenic exotoxins A and B, respectively (25, 26). We use the The cut-off value was determined by preliminary experiments term erythrogenic toxin because we have routinely assayed bio- using serial dilutions of high-titered human sera. logic activity of the toxins by skin reaction in rabbits. Erythro- Fractionation of human sera by protein A Sepharose column. genic and mitogenic activities of the toxins have not until now A total of 50 to 300 pL of human serum or Ig fraction prepared been separable. Doses of the toxin preparations were shown in by ammmonium sulfate precipitation at 50% saturation was terms of protein concentration. diluted to 1 mL with PBS, applied to 2.5 mL of swollen protein Animals. Adult JW-NIBS strain rabbits (The Nippon Institute A Sepharose packed in a 5-mL syringe, and passed through the for Biological Sciences, Tokyo, Japan) of either sex were used. column by addition of PBS under monitoring by UV absorption. Rabbit lymphocyte culture. A modification of the previous IgG was eluted by glycine-HC1 buffer. Both the effluent and the methods (27, 28) was used. Peripheral blood from ear artery was eluate fractions were concentrated by use of PMlO filter (Ami- defibrinated by glass beads in a flask, mixed with an equal volume con, Lexington, MA), diluted with PBS when necessary, and of 1% polyvinyl alcohol in PBS containing carbonyl iron powder brought to the same volume to 20-fold dilution of the starting (20 mg/mL) after separation of serum, and incubated at 37°C specimen of serum.
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